When a blood vessel is cut, the coagulation protease, thrombin, quickly activates platelets and fibrinogen to form a hemostatic plug. Thrombin activation of human platelets is mediated by dual protease-activated receptors--a high affinity PAR1 thrombin receptor and a low affinity PAR4 thrombin receptor. PAR1 and PAR4 are specifically tuned to be cleaved at very different rates which gives rise to their respective roles in the initiation and propagation phases of platelet aggregation. Chronic stimulation of PAR1 in the pro-thrombotic milieu of an atherosclerotic plaque has also been implicated in smooth muscle cell proliferation and in restenosis following acute coronary interventions in humans and animal model systems. More recently, it has been shown that PAR1 expression is upregulated in malignant cells and controls the invasion of epithelial-derived breast cancer cells and the metastasis of melanoma cells. Thrombin exerts these diverse cellular effects by cleaving the PAR exodomains to create a new N-terminus that binds to the extracellular loops of the receptor in an unusual intramolecular liganding mode. The first goal of these studies is to determine the underlying functional and structural basis of the differences in intramoleular liganding of PAR1 in extracellular loops e2-e4 and PAR4 in the N-terminal exodomain using NMR and biochemical approaches. We will also determine the structure of a non-cleavable PAR1 exodomain 'substrate' in complex with thrombin. Our recent kinetic and NMR structural studies demonstrated that the cleaved PAR1 exodomain product retains high-affinity binding to exosite I of thrombin via its hirudin-like sequence but leaves the active site and exosite II (heparin-binding site) of thrombin freely accessible to other large macromolecules such as PAR4 and GPIb. The second aim focuses on the thrombin binding and cleavage reactions occurring with intact PAR1 and PAR4 on the surface of platelets and mammalian cells. We will determine whether platelet PAR1 and the GPIb-IX-V von Willebrand factor receptor serve as thrombin-binding cofactors for PAR4. The formation of hetero- and homo-dimers of PAR1 and PAR4 will be detected by fluorescence resonance energy transfer (FRET) of PAR-YFP and PAR-CFP pairs, and by biochemical analysis of epitope-tagged receptors. In the third aim, we will determine what role tissue factor plays in the in situ generation of thrombin and cleavage of PARs on the surface of cancer cells. We anticipate that these studies will lead to the identification of novel intra- and inter-molecular interactions between the various thrombin receptors and accessory proteins that may be critical for the regulation of thrombin signaling during hemostasis, thrombosis, and metastasis.